PROJECT SUMMARY/ABSTRACT A critical barrier to treating Type 1 Diabetes (T1D), an autoimmune disease in which the islet beta cells are destroyed by immune cells, is understanding the heterogeneity of disease. One source of immune heterogeneity recently identified across recent onset T1D subjects is the presence of partially exhausted CD8 T cells in subjects with beneficial response to anti-CD3 therapy (Long, et.al Science Immunology). Likewise, subjects with slower disease progression have great frequencies of islet-specific CD8 T cells with more features of exhaustion. Yet, the mechanisms underlying exhaustion in T1D are not well understood. By contrast, in chronic viral settings it is well established that the IL-2/IL-15 axis is involved in differentiation and persistence and CD8 exhaustion: increased IL-2 availability prevents differentiation, while response to IL-2/IL- 15 is required for persistence. In T1D, we published seminal studies along with others that link alterations in the IL-2/IL-15 axis with T1D. Here, we find that T1D subjects with lower frequencies of exhausted CD8 T cells have higher levels of CD4 IL-2 production and lower levels of IL-15 response in CD8 exhausted cells. Thus, IL- 2/IL-15 defects associated with T1D present a unique opportunity to elucidate the role of IL-2 on CD8 exhaustion. We hypothesize that alterations in the IL-2/IL-15 axis in T1D lead to decreased differentiation into islet-specific exhausted CD8 T cells and reduced persistence. We will test this hypothesis in two focused aims using samples from well curated human cohorts of established T1D, pre- clinical T1D and healthy control (HC) subjects, and multi-dimensional single cell approaches including mass cytometry (CyTOF), RNA-seq and ATAC-seq combined with innovative systems immunology approaches. In Aim 1, we propose to elucidate mechanisms of increased CD4 IL2 production that lead to reduced exhaustion at different stages of disease. In Aim 2, we will elucidate mechanisms of reduced IL-12/IL-15 response in exhausted CD8 T cells that results in limited persistence of exhaustion. Successful completion of these aims will 1) advance our understanding of CD8 T cell exhaustion as an understudied form of tolerance in T1D, 2) improve our understanding of immune factors contributing to T1D disease progression, and 3) provide the foundation for the development of therapies designed to promote exhaustion.